Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as a model system for cell membranes. However the study of proteo-membrane interactions in this system is limited as the incorporation of integral and lipid-anchored proteins into GUVs remains challenging. Here, we present a simple generic method to incorporate proteins into GUVs. The basic principle is to break proteo-liposomes by an osmotic shock. They subsequently reseal into larger vesicles which, if necessary, can endure the same to obtain even bigger proteo-GUVs. This process does not require specific lipids or reagents, works under physiological conditions with high concentrations of protein, the proteins remains functional after incorporation and the resulting proteo-GUVs can be micromanipulated. Moreover, our protocol is valid for a wide range of protein substrates. We have successfully reconstituted three structurally different proteins, two trans-membrane proteins, TolC and the neuronal t-SNARE, and one lipid-anchored peripheral protein, GABARAP-Like 1 (GL1). In each case, we verified that the protein remains active after incorporation and in their correctly folded state. We also measured their mobility by performing diffusion measurements via fluorescence recovery after photobleaching (FRAP) experiments on micromanipulated single GUVs. The diffusion coefficients are in agreement with previous data.

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The Atg8/LC3/GABARAP protein family has been implicated in membrane remodeling events on the growing autophagosome. In particular, each of these proteins can form a protein-lipid conjugate that has been shown in vitro to drive liposome aggregation and in some cases membrane fusion. Furthermore, yeast Atg8 has been described as a curvature sensing protein, through its natural capacity to concentrate on highly curved membranes. A key advance with yeast Atg8, was the introduction of Giant Unilamellar Vesicles (GUVs) as an in vitro support that could allow membrane deformation and tethering to be observed by simple microscopy. Further, micromanipulation of an individual GUV could be used to create local areas of curvature to follow Atg8 partitioning. Here, we use a recently developed method to decorate GUVs with the mammalian Atg8 protein GABARAPL1 and establish the generality of the observations made on yeast Atg8. Then we apply double micromanipulation, the capture and positioning of two independently prepared GUVs, to test elements of the mechanism, speed and reversibility of mammalian Atg8 protein-mediated tethering. We find that the membranes adhere through GABARAPL1/GABARAPL1 homotypic trans-interactions. On a single membrane with two regions with significantly different curvatures we observed that the regions of higher curvature can be enriched up to 10 times in GABARAPL1 compared to the planar regions. This approach has the potential to allow the formation and study of specific topographically-controlled interfaces involving Atg8-proteins and their targets on apposing membranes.

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